Wow, this seems like a very cool machine. It does seem prohibitively expensive though for a class to use just a couple times. The cost could probably be justified in a lab that needs to separate cells a lot.

Variations of Protocol 15.1 include position of cells, other media, anduse of markerbeads. Marker beads can be used to establish different density areas of the gradient.

Isopyknic sedimentation is faster and gives higher yields than velocity sedimentation. It shouldbe used when there are clear differences in density.

15.2

Cell Size and Sedimentation Velocity

A relationship exists between particle size and sedimentation rate, and is givenapproximately by:

v

r^2/4

15.2.1 Unit Gravity Sedimentation

If you layer cells over a serum gradient, the cells will settle by

the equation given above.There is a caveat, unit gravity sedimentation is limited to smaller numbers of cells.

15.2.2 Centrifugal Elutriation

A centrifugal elutriator increases sedimentation rate by separating cells in a centrifugespecifically made for separation. Basically, cells are pumped into a separation chamber, whichpushed them to the outer edge. Medium is pumped through to balance the sedimentation rate ofthe cells. Because of the differences among cells, sedimentation occurs at different rates. Thetapered shape of the chamber means there is a gradient of flow rates.

Wow, this seems like a very cool machine. It does seem prohibitively expensive though for a classto use just a couple times. The cost could probably be justified in a lab that needs to separatecells a lot.

15.3 Antibody-based Techniques

Antibody-based techniques depend on antibody specific binding to an epitope.

15.3.1 Immune Panning

Immune panning: attaching cells to dishes coated with antibodies. The cellsthat aretargeted by the antibodies will attach at the bottom of the cell.

15.3.2 Magnetic Sorting

This technique uses antibodies against a cell surface epitope that are conjugated to micro-or ferritin beads. The cells are mixed with the beads and placed in a magnetic field. The cellsattached to the beads will separate.

This technique uses a laser beam so that cells will scatter light. A flow cytometermeasures photomultipliers. A fluorescence-activated cell sorter will use the emission from eachcell to sort it into sample tubes or waste container.

Iremember lasers being used in plant tissue culture techniques. It’s cool that they have so manyapplications.

Other markers include chromosomal abnormalities, MHC group antigens, and DNAfingerprinting.

16.3.4 Transformation

Transformation is such a large topic that it is discussed in Ch 17.

16.4 Cell Morphology

Monitoring morphology is the easiest way to identify cells, but has some deficiencies.One of which is how cell morphology changes depending on culture conditions. Despite theseshortcomings, frequent observations of cultures are more useful than occasional stains. Recallfrom chapter 13 that unhealthy cells will become granular and show vacuolation around thenucleus.

16.4.1Microscopy

The inverted microscope is often used incorrectly. Phase-contrast increases contrast ofunstained cells.

Protocol 16.1 outlines how to use an inverted microscope. Place the culture on the microscopestage, choose the correct optics. Focus and center.

I would have assumed that using a microscope is a basic task, but I suppose it doesn’t hurt tocover the basics.

16.4.2 Staining

Giemsa is one way to easily prepare a stained culture.

Protocol 16.2 shows how to stain with Giemsa: fix culture inmethanol, stain with Geimsa, anddilute to 1:10. Wash and examine.

Fingerprinting is also useful in TC in that it can exclude cross-contamination and confirmidentity.

16.6.3 DNA Profiling

DNA profiling is only used in human now and is based on the fact that short tandemrepeats of microsatellite sequences have been quantified.

Protocol 16.9 outlines how to perform DNA profiling.

16.7 RNA and Protein Expression

A Northern blot is a way to analyze gene expression. When extracts are run are 2-D gels,they produce a wealth ofinformation difficult to interpret. Using a computer to scan the gelshelps to produce the data efficiently.

16.8 Enzyme Activity

Some, but not many, enzymes will be expressedin vitro. If using marker enzymes,compare the uninduced and induced levels with controls.

16.8.1 Isoenzymes

Enzyme activity can also be studied by comparing cell strains among species.

16.8.2 Isoenzyme Electrophoresis with Authentikit

Authentikit screens 6 cell lines for 7 genetic makers.

16.9.1 Immunostaining

Antibody location can be determined through immunostaining. This method uses anantibody conjugated to a fluorochrome, or depositing a product conjugated to an antibody. It canbe direct, which is when the antibody is conjugated to the fluorochrome/enzyme and stainsdirectly. Or it can be direct,in which the primary antibody binds to the antigen. Then there is asecond antibody against the Ig of the first antibody. The second antibody is conjugated withfluorochrome.

I wonder why indirect is more common. It sound like it would be more complicated to have twoantibodies instead of one.

16.9.2 Immunoanalysis

Assaying quantifies marker and protein expression. They can be done through kits.